The present invention is related to the production of shikimic acid and more specifically, to methods of producing shikimic acid from bioconversion of a carbon source.
Shikimic acid is an attractive chiral synthon with its highly functionalized, six-membered carbocyclic ring and multiple asymmetric centers. A metabolic intermediate of aromatic amino acid biosynthesis, shikimic acid has emerged as an essential chiral starting material in the synthesis of neuraminidase inhibitors effective in the treatment of influenza. Kim. C.U. et al., J. Am. Chem. Soc. 119:681 (1997); Rohloff, J.C. et al., J. Org. Chem. 63:4545 (1998). Chiral, as well as aromatic chemicals, can also be synthesized from shikimic acid. For example, acid catalyzed dehydration of shikimic acid affords p-hydroxybenzoic acid (Eykmann, J.F., Ber. Dtch. Chem. Ges. 24:1278 (1891)). p-Hydroxybenzoic acid, which has an annual production of 7xc3x97106 kg, is the key precursor to parabens and a monomer used in the synthesis of liquid crystal polymers. Shikimic acid has also recently been used as the starting point for synthesis of a large combinatorial library of molecules. Tan, D.S. et al., J. Am. Chem. Soc. 120:8565 (1998).
Shikimic acid is obtained via tedious multi-step isolation procedures from plants. Unfortunately, current isolation of shikimic acid from the fruit of Illicium plants (Haslem, E., Shikimic Acid: Metabolism and Metabolites, Wiley and Sons, New York, pp. 40-42 (1993)) precludes its use in kilogram-level synthesis.
Therefore, it would be desirable to provide a method to produce large quantities of shikimic acid. It would also be desirable if such a method were cost-efficient, using inexpensive starting materials. It would further be desirable if the method employed non-toxic compounds and was environmentally benign.
A bioengineered synthesis scheme for production of shikimic acid from a carbon source is provided. In one embodiment, the bioconversion methods of the present invention comprise the microbe-catalyzed conversion of a carbon source to shikimic acid. The method comprises selecting a host cell, introducing into the host cell the ability to convert the carbon source to shikimic acid in the host cell, impeding in a pathway of the host cell the conversion of shikimic acid to shikimate-3-phosphate and culturing the host cell in the carbon source. The methods further comprise contolling the molar ratio of shikimic acid to quinic acid during culturing. As shown in the synthesis scheme of FIG. 1, the microbe-catalyzed conversion step of the present invention requires four enzymes which may be provided by a recombinant microbe. In a preferred embodiment, the recombinant microbe is Escherichia coli designed to cause reduction of 3-dehydroshikimate to shikimic acid and to inhibit any further conversion of shikimic acid along the aromatic amino acid biosynthetic pathway.
In another embodiment, methods are provided for increasing the production of shikimic acid with the recombinant microbes of the present invention. In a preferred embodiment, the methods comprise blocking the phosphoenolpyruvate carbohydrate phosphotransferase system of the host cell and introducing into the host cell the ability to transport glucose into the cell. In an alternate preferred embodiment, the methods comprise introducing into the host cell the ability to convert pyruvate to phosphoenolpyruvate. In a further preferred embodiment, the host cell is E. coli B. Production of shikimic acid in the methods of the present invention is increased when this strain of E. coli is used as the host cell.
The biocatalytic synthesis method for shikimic acid provided herein, is believed to be environmentally benign, economically attractive, and utilizes abundant renewable sources as a starting material.
Additional objects, advantages, and features of the present invention will become apparent from the following description and appended claims, taken in conjunction with the accompanying drawing.